Azacytidine analogues and uses thereof

ABSTRACT

The present invention is directed toward compounds of Formula (I) as follows: wherein, R is H, R5C(O), R5CH2OC(O), or R5CH2NHC(O); R1 is where the crossing dashed line illustrates the bond formed joining R1 to the molecule of Formula (I); R2 and R3 are independently OH or H, provided that R2 and R3 are not simultaneously OH; R4 is H, R5C(O), R5CH2OC(O), or R5CH2NHC(O), provided that R and R4 are not simultaneously H; and R5 has the general formula: CH3—(CH2)n—(CH═CH—CH2)m—CH═CH—(CH2)k—; k is an integer from 0 to 7; m is an integer from 0 to 2; and n is an integer from 0 to 10, or a pharmaceutical salt thereof. Methods of making and using these compounds are also disclosed.

This application claims benefit of U.S. Provisional Patent ApplicationSer. No. 60/975,437, filed Sep. 26, 2007, which is hereby incorporatedby reference in its entirety.

FIELD OF THE INVENTION

This invention relates to azacytidine analogues and uses thereof.

BACKGROUND OF THE INVENTION

Nucleoside analogues, the derivatives of the natural nucleosides foundas building blocks of DNA and RNA, are effective in the clinicaltreatment of human cancer or viral diseases, although in the early yearssuch compounds were evaluated as anti-tuberculosis agents. Suchcompounds have been registered in the market for more than 40 years, andapproximately 35 products are currently in daily use. The naturalnucleosides illustrated in Table 1 below, are constructed from twoclasses of nitrogen bases, i.e. the purines (exemplified by adenine andguanine) and the pyrimidines (exemplified by thymine, uracil, andcytosine) and from the monosaccharide ribose or deoxyribose.

TABLE 1 Purines

Pyrimidines

Monosaccharides

The natural nucleosides all exist in the so called β-D configuration asillustrated in the Formula A below. The nitrogen base and thehydroxy-methyl side chain on the sugar ring are both on the same side(cis) of the plane of the sugar ring.

In order to obtain nucleoside derivatives with anticancer or antiviralactivity, chemical modifications in either the nitrogen base and/or themonosaccharide have been performed. For instance in the nitrogen base,the addition of halogen atoms or other functional groups, insertion ofadditional nitrogen atoms or a stereochemical change in themonosaccharide ring from ribose to arabinose or removal of the hydroxylgroup to deoxyribose may lead to products with a potential therapeuticbenefit. In many products, the monosaccharide ring is conserved, whilein others, the sugar ring has been changed into a chain. The nucleosideanalogues are small molecules with fair to excellent aqueous solubility.

The extensive research and development effort put into the area ofnucleoside analogues due to the worldwide AIDS epidemic bolstered thebasic knowledge and understanding of mechanism of action, alterations inactivity profile due to chemical modifications etc, are also relevant tothe field of cancer treatment.

A general weakness with many drugs, including nucleoside analogues, islow activity and inferior specificity for treatment of the actualdisease in question. Some of these problems may be related to theinherent activity of the drug substance itself, some may be related tocertain resistance mechanisms (either inherent in the patient oracquired during treatment e.g. multiple drug resistance (MDR) in cancertreatment). Some problems may be related to certain inferior transportor cellular uptake and activation mechanisms. Some problems may berelated to rapid inactivation and/or excretion of the drug.

The efficacy of nucleoside analogues depends on a large extent on theirability to mimic natural nucleosides, thus interacting with viral and/orcellular enzymes and interfering with or inhibiting critical processesin the metabolism of nucleic acids. In order to exert their antiviral oranti-cancer activity, the nucleoside analogues have to be transformed,via their mono- and di-phosphates, into their correspondingtri-phosphates through the action of viral and/or cellular kinases. As ageneral rule, the tri-phosphate is the active agent, but for someproducts, e.g. gemcitabine, even the di-phosphate may exert a clinicallysignificant effect.

In order to reach the diseased, cancerous or virus infected cells ortissues, following either enteral or parenteral administration, thenucleoside analogues should have favorable pharmacokineticcharacteristics. In addition to rapid excretion of the administereddrug, many nucleoside analogues may be deactivated both in the bloodstream and in tissues. For instance, cytosine derivatives, even at themono-phosphate level, may be rapidly deaminated through the action of aclass of enzymes called deaminases, to the inactive uracil analogue. Thecellular uptake and thus good therapeutic efficacy of many nucleosideanalogues strongly depend on membrane bound nucleoside transportproteins (called concentrative and equilibrative nucleosidetransporters). Hence, compounds that do not rely on such specific uptakemechanisms are sought. Yet another activity limiting factor,particularly within the anti-cancer field, are the cellular repairmechanisms. When an anti-cancer nucleoside analogue mono-phosphate isincorporated into the cellular DNA, it should not be removed from thecancer cell DNA due to the exonuclease activity linked to the p53protein. However, removal of a nucleoside analogue from the DNA of ahealthy cell is favorable in order to limit the side effects of thedrug.

Over the years, many nucleoside analogues have been developed that to alarge extent overcome some or many of the activity limiting features. Asan example, acyclovir (ACV) can be given to illustrate a compound withgreat specificity. The ACV-mono-phosphate can only be formed by viralkinases meaning that ACV cannot be activated in uninfected cells.Despite this fact, ACV is not a particularly active product. In order tocircumvent the often rate limiting step in the activation of anucleoside analogue, the intracellular formation of the nucleosideanalogue mono-phosphate, several phosphonates, such as cidofovir or evenmono-phosphate products, have been developed. In order to facilitateoral uptake or to secure a favorable drug disposition in the body,particular prodrugs such as Hepsera have been made.

In addition to the structural changes made to nucleoside analogues tofacilitate enhanced clinical utility, further modifications have beenmade to improve the activity. There are several examples of modifiednucleoside analogues resulting from the addition of lipid moieties (U.S.Pat. Nos. 6,153,594, 6,548,486, 6,316,425, and 6,384,019; EuropeanPatent Application Nos. EP-A-56265 and EP-A-393920; and WO 99/26958).This can be achieved by the linking of fatty acids through, forinstance, an ester, amide, carbonate, or carbamate bond. More elaborateproducts can be made, such as phospholipid derivatives of the nucleosideanalogues. See Eur J Pharm Sci 11b Suppl 2: 15-27 (2000); EuropeanPatent No. 545966; Canadian Patent No. 2468099; and U.S. Pat. Nos.6,372,725 and 6,670,341. Such analogues are described to have antiviralactivity that is particularly suitable for the therapy and prophylaxisof infections caused by DNA, RNA, or retroviruses. They are also suitedfor treatment of malignant tumours. The nucleoside analogue lipidderivatives may serve several purposes. They may be regarded as aprodrug that is not a substrate for deaminases, thereby protecting thenucleoside analogues from deactivation during transport in thebloodstream. The lipid derivatives may also be more efficientlytransported across the cellular membrane, resulting in enhancedintracellular concentration of the nucleoside analogue. Lipidderivatives may also be more suited for use in dermal preparations, oralproducts (see U.S. Pat. No. 6,576,636 and WO 01/18013), or particularformulations such as liposomes (see U.S. Pat. No. 5,223,263) designedfor tumor targeting.

It has been demonstrated that for nucleoside analogues with a conservedβ-D configuration of the monosaccharide ring, or for nucleosideanalogues with a non-cyclic side chain, the antiviral or anticanceractivity can be most efficiently improved through the formation of lipidderivatives of mono-unsaturated ω-9 C18 and C20 fatty acids. SeeAntimicrobial Agents and Chemotherapy, Vol., 53-61 (1999); CancerResearch 59: 2944-2949 (1999); Gene Therapy, 5: 419-426 (1998);Antiviral Research, 45: 157-167 (2000); and Biochemical Pharmacology,67: 503-511 (2004). The preferred mono-unsaturated derivatives are notonly more active than the poly-unsaturated counterparts but are morecrystalline and chemically stable towards oxidation of the lipid chain.Hence, they are more favorable compounds from a chemical andpharmaceutical manufacturing point of view. It has also demonstratedthat the mono-unsaturated ω-9 C18 and C20 fatty acids are suited forimprovement of the therapeutic activity of a large number ofnon-nucleoside biologically active compounds (see European Patent No.0977725).

A relatively new subgroup of nucleoside analogues are the so calledaza-C derivatives. In this class of compounds, the CH group in the 5position in the pyrimidine base is exchanged with a nitrogen atom asshown in Formula B below.

Tumor suppressor genes that have been silenced by aberrant DNAmethylation are potential targets for reactivation by these novelchemotherapeutic agents. The potent inhibitors of DNA methylation andantileukemic agents, aza-cytidine and 5-aza-2′-deoxycytidine derivatives(5-aza-C, 5-aza-CdR, Decitabine), can reactivate silent tumor suppressorgenes. At high concentrations, the compounds are cytotoxic, but at lowerconcentrations the hypomethylation leads to differentiation of celllines. The compounds requires metabolic activation by deoxycytidinekinase, and produces an inhibition of DNA methyltransferase. Onehindrance to the curative potential of these derivatives is their rapidin vivo inactivation by cytidine deaminase (CD). The instability inaqueous solutions as well as their side effect profiles have limitedclinical activity.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed toward a compoundaccording to Formula (I)

wherein R is H, R₅C(O), R₅CH₂OC(O), or R₅CH₂NHC(O), R₁ is

where the crossing dashed line illustrates the bond formed joining R₁ tothe molecule of Formula (I), R₂ and R₃ are independently OH or H,provided that R₂ and R₃ are not simultaneously OH, R₄ is H, R₅C(O),R₅CH₂OC(O), or R₅CH₂NHC(O), provided that R and R₄ are notsimultaneously H, R₅ has the general formula:

-   CH₃—(CH₂)—(CH═CH—CH₂)_(m)—CH═CH—(CH₂)_(k)—; k is an integer from 0    to 7; m is an integer from 0 to 2; and n is an integer from 0 to 10,    or a pharmaceutical salt thereof.

Another aspect of the present invention is directed toward apharmaceutical composition comprising the compound of Formula (I) and apharmaceutical excipient, diluent, and/or carrier.

A further aspect of the present invention is directed toward a method oftreating a subject for a neoplastic condition. The method includesselecting a subject with a neoplastic condition and administering to thesubject a compound of Formula (I), as described above, or apharmaceutical salt thereof, under conditions effective to treat theneoplastic condition in the subject.

A further aspect of the present invention is directed toward a method oftreating a subject for an inflammatory condition. The method includesselecting a subject with an inflammatory condition and administering tothe subject a compound of Formula (I), as described above, or apharmaceutical salt thereof, under conditions effective to treat theinflammatory condition in the subject.

The instability of Aza-C in buffer and plasma is well known (see Israiliet al., Cancer Research 36, 1453-1461 (1976); Rudek et al., J ClinOncol, 23:17, 3906-3911 (2005); Rustum et al., J Chromat, 421:12, 387-91(1987); Zhao et al., J Chromat B, 813, 81-88 (2004), which are herebyincorporated by reference in their entirety). An average terminalhalf-life of 1.50±2.30 hours in clinical plasma samples has beenreported for Aza-C (see Rudek et al., J Clin Oncol, 23:17, 3906-3911(2005), which is hereby incorporated by reference in its entirety). Invitro, a 20% loss of Aza-C even at −60° C. is noted after 4.5 daysstorage and a 10% loss within 0.5 hours when stored at room temperature(see Zhao et al., J Chromat B, 813, 81-88 (2004), which is herebyincorporated by reference in its entirety). The prime instability ofAza-C is thought to be due to a rapid (first step being reversible) ringopening of the 5-Aza-pyrimidine ring with a subsequent elimination offormic acid (see Chan et al., J Pharma Sci, 68; 7, 807-12 (1979), whichis hereby incorporated by reference in its entirety). Other degradationpathways are thought to be deamination of the position 4 amino group andhydrolysis of the glycoside bond to give D-ribose and 5-azacytosine. Ithas been surprisingly found that the preferred Aza-C lipid derivativeshave a significantly better plasma stability profile than Aza-C itself.The compounds are stable (percent remaining of initial ≧94%) in blankhuman plasma matrix at room temperature for at least 4 hours under theexperimental conditions, and no significant degradation products wereobserved in the post-extract supernatant after precipitation of plasmaproteins. The plasma stability of the preferred lipid compounds havebeen examined further when stored at 37° C. It is shown that thering-opening of the Aza-moiety or other degradation of the compound issignificantly reduced when the lipid side chain is attached to Aza-C.

The rapid degradation of Aza-C is a drawback for clinical use of Aza-C.The enhanced plasma stability of the lipid derivatives over Aza-C itselfmay give both a high and sustained patient plasma level of the lipidderivative. This may lead to a better tissue/organ/tumor distributionand cellular exposure to and uptake of the drug than for Aza-C itself inthe first hand, and subsequently better tumor cell DNA exposure to Aza-Cafter intracellular hydrolysis of the Aza-C-5′-ester bond.

Embodiments of the present invention create, through the modification ofazacytidine and deoxycytidine (e.g., 5-aza-2′-deoxycytidine), novelmolecules with surprisingly different properties compared to azacytidineand deoxycytidine (e.g., 5-aza-2′-deoxycytidine). This creates a seriesof compounds with activity that extends well beyond the anti-canceractivity of azacytidine and deoxycytidine (e.g., 5-aza-2′-deoxycytidine)which is limited to hematologic malignancies. These novel compounds haveanti-cancer efficacy against a broad array of solid tumors includingbreast and cervical cancer. The compounds are also surprisingly activeagainst cancers which are treatment resistant and thus can offer atherapeutic advantage in solid tumors where current treatment choicesare limited. Embodiments of the present invention have therapeutic usesto treat cancers where options and efficacy remain limited and fulfillan unmet need.

These compounds exhibit an earlier onset of activity after limitedexposure and, therefore, can be effective after only a short duration oftreatment in the clinical setting. This would translate into shorter,less frequent treatment exposure and a reduction in drug-relatedtoxicities compared to the parent drugs. This would provide for anenhanced therapeutic index.

The alteration in the structure with the addition of the lipid (includesboth esters, amides, carbamates and carbonates) component conserves theazole cytidine ring and thus the effects of the molecule on epigeneticmechanisms. Epigenetic modulation offers an important mechanism foraltering gene expression in cancer and inflammation. These novelcompounds have activity at lower concentrations than azacytidine and,thus, are more potent. These compounds with an altered spectrum ofactivity can modulate epigenetic targets in solid tumors andinflammatory diseases.

Epigenetic mechanisms are important in pro-inflammatory states whichinclude, but are not exclusive to, inflammatory states of the lung,connective tissues, gastro-intestinal tract and vasculature. Thesecompounds, by targeting epigenetic mechanisms, can reduce or reverse theinflammatory processes responsible for these diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a time profile for cytotoxic activity forAza-c and 5-Aza-C-5′-petroselinic acid.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention is directed toward a compoundaccording to Formula (I)

wherein R is H, R₅C(O), R₅CH₂OC(O), or R₅CH₂NHC(O), R₁ is

where the crossing dashed line illustrates the bond formed joining R₁ tothe molecule of Formula (I), R₂ and R₃ are independently OH or H,provided that R₂ and R₃ are not simultaneously OH, R₄ is H, R₅C(O),R₅CH₂OC(O), or R₅CH₂NHC(O), provided that R and R₄ are notsimultaneously H, and R₅ has the general formula:

-   CH₃—(CH₂)—(CH═CH—CH₂)_(m)—CH═CH—(CH₂)_(k)—; k is an integer from 0    to 7; m is an integer from 0 to 2; and n is an integer from 0 to 10,    or a pharmaceutical salt thereof.

In preferred embodiments, k is 4 and n is 10. In certain embodiments, R₁is

where the crossing dashed line illustrates the bond formed joining R₁ tothe molecule of Formula (I). In some embodiments, R₄ may be H. Incertain embodiments, R is R₅C(O), k is 4, m is 0, n is 10, R₂ is H, andR₃ is OH, and R₄ is H.

A broader aspect of the present invention is directed toward a compoundaccording to Formula (I)′

wherein R is H, R₅C(O), R₅CH₂OC(O), or R₅CH₂NHC(O), R₁ is

where the crossing dashed line illustrates the bond formed joining R₁ tothe molecule of Formula (I)′, R₂ and R₃ are independently OH or H,provided that R₂ and R₃ are not simultaneously OH, R₄ is H, R₅C(O),R₅CH₂OC(O), or R₅CH₂NHC(O), provided that R and R₄ are notsimultaneously H, and R₅ is a C₃-C₂₆ alkenyl, or a pharmaceutical saltthereof.

In a preferred embodiment of the compound according to Formula (I)′, kis 4 and n is 10. In certain embodiments, R₁ is

where the crossing dashed line illustrates the bond formed joining R₁ tothe molecule of Formula (I)′. In some embodiments, R₄ may be H. In otherembodiments, R is R₅C(O), k is 4, m is 0, n is 10, R₂ is H, and R₃ isOH. In certain embodiments R₅ is a C₉-C₂₆ alkenyl.

Another aspect of the present invention is directed toward apharmaceutical composition comprising the compound of Formula (I) and apharmaceutical excipient, diluent, and/or carrier.

Agents of the present invention can be administered orally,parenterally, for example, subcutaneously, intravenously,intramuscularly, intraperitoneally, by intranasal instillation, or byapplication to mucous membranes, such as, that of the nose, throat, andbronchial tubes. They may be administered alone or with suitablepharmaceutical carriers, and can be in solid or liquid form such as,tablets, capsules, powders, solutions, suspensions, or emulsions.

The active agents of the present invention may be orally administered,for example, with an inert diluent, or with an assimilable ediblecarrier, or they may be enclosed in hard or soft shell capsules, or theymay be compressed into tablets, or they may be incorporated directlywith the food of the diet. For oral therapeutic administration, theseactive agents may be incorporated with excipients and used in the formof tablets, capsules, elixirs, suspensions, syrups, and the like. Suchcompositions and preparations should contain at least 0.1% of activeagent. The percentage of the agent in these compositions may, of course,be varied and may conveniently be between about 2% to about 60% of theweight of the unit. The amount of active agent in such therapeuticallyuseful compositions is such that a suitable dosage will be obtained.Preferred compositions according to the present invention are preparedso that an oral dosage unit contains between about 1 and 250 mg ofactive agent.

The tablets, capsules, and the like may also contain a binder such asgum tragacanth, acacia, corn starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, alginic acid; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, lactose, or saccharin. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier, such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar, or both. A syrup may contain, in addition to theactive ingredient, sucrose as a sweetening agent, methyl andpropylparabens as preservatives, a dye, and flavoring such as cherry ororange flavor.

These active agents may also be administered parenterally. Solutions orsuspensions of these active agents can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof in oils. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solution, and glycols such as, propylene glycol or polyethyleneglycol, are preferred liquid carriers, particularly for injectablesolutions. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

The agents of the present invention may also be administered directly tothe airways in the form of an aerosol. For use as aerosols, the agentsof the present invention in solution or suspension may be packaged in apressurized aerosol container together with suitable propellants, forexample, hydrocarbon propellants like propane, butane, or isobutane withconventional adjuvants. The materials of the present invention also maybe administered in a non-pressurized form such as in a nebulizer oratomizer.

A further aspect of the present invention is directed toward a method oftreating a subject for a neoplastic condition. The method includesselecting a subject with a neoplastic condition and administering to thesubject a compound of Formula (I), as described above, or apharmaceutical salt thereof, under conditions effective to treat theneoplastic condition in the subject.

In certain embodiments, the neoplastic condition is a cancerous disease.The cancerous disease may be a solid tumor or a hematological cancer ormalignancy. The cancerous disease may be leukemia, lymphoma, multiplemyeloma, or myelodysplastic syndrome.

In certain embodiments, the solid tumor may be a cancer of a tissue suchas breast, ovary, prostate, brain, bladder, and lung tissues.

A further aspect of the present invention is directed toward a method oftreating a subject for an inflammatory condition. The method includesselecting a subject with a an inflammatory condition and administeringto the subject a compound of Formula (I), as described above, or apharmaceutical salt thereof, under conditions effective to treat theinflammatory condition in the subject.

In certain embodiments, the inflammatory condition is an inflammatorystate of the lung, connective tissue, gastro-intestinal tract, orvasculature.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

EXAMPLES Example 1 Reagents, Cell Lines, and Cell Culture

Cell proliferation reagent WST-1 was obtained from Roche Applied Science(Manheim, Germany), PI and Annexin V-FITC apoptosis kit were purchasedfrom BD Biosciences, Palo Alto, Calif., 5-azacytidine (5-AzaC), ethidiumbromide (EB), acridine orange (AO), nitro blue tetrazolium (NBT),phorbol 12-myristate 13-acetate (TPA) were purchased from Sigma ChemicalCo (St. Louis, Mo.).

Human promyelocytic leukemia cell lines HL60, human histiocytic lymphomaU937, human chronic myelogenous leukemia K562, human acute T cellJurkat, breast adenocarcinoma MCF-7, urinary bladder carcinoma 5637,prostate carcinoma DU-145 were purchased from American Type CultureCollection. All cell lines except Jurkat were maintained in RPMI 1640medium (Gibco, Glasgow, UK) supplemented with 10% heat-inactivated fetalcalf serum (FCS), 100 U/ml of penicillin, and 100 mg/ml streptomycin, inan atmosphere of 5% CO₂ at 37° C. Jurkat cells were cultured in RPMI1640 medium supplemented with 1.5 g/L sodium bicarbonate, 4.5 g/Lglucose, 10 mM sodium pyruvate, and 10% FCS, 100 U/ml of penicillin, and100 mg/ml streptomycin.

Example 2 Cytotoxicity Assay

The cytotoxicity of 5-azacytidine lipid was determined by calorimetricassay based on the cleavage of the tetrazolium salt WST-1(4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate) by mitochondrial dehydrogenases in viable cells. Cells wereseeded at an initial concentration of 1×10⁶/ml (HL60 cells) or1.25×10⁵/ml (U937, K562 and Jurkat) in medium with or without variousconcentrations of 5-azacytidine lipid in a 96-well flat bottommicro-plates and cultured for 24 to 72 hours. MCF-7, DU-145, and 5637cells (1×10⁴/ml) were plated and allowed to adhere and spread for 24hours. The various concentrations of 5-azacytidine lipid were added andcultures were maintained for an additional 24 to 72 hours. Cultures wereincubated with WST-1 reagent for 1 hour. The production of formazan wasmeasured by a microplate reader (Bio-Tek Instruments, Elx 800) at 450 nmwith a reference wavelength of 650 nm. Growth inhibition was determinedas compared to untreated cells (%). IC 50 values were calculated usingCalcuSyn software (Biosoft).

Example 3 Quantitation of Apoptotic Cells

Apoptotic cells were defined using morphological criteria andfluorescence-activated cell sorting (FACS) after staining with AnnexinV-FITC. For morphologic analysis, 1 μl of stock solution containing 100μg/ml AO and 100 μg/ml EB was added to 25 μl cells suspension. Theapoptotic cells and apoptotic bodies were analyzed with the aid of afluorescence microscopy. The percentage of apoptotic cells wascalculated after counting total 300 cells. For FACS analysis, 2×105 to5×106 cells were washed with PBS and then labeled with Annexin V-FICSand propidium iodide (PI) in medium-binding reagent according to theAnnexin V-FITC apoptosis detection kit instruction provided by themanufacturer. Fluorescent signals of FITC and PI were detected,respectively, at 518 nm and at 620 nm on FACSCAN (Becton Dickinson, SanJose, Calif.). The log of Annexin V-FITC fluorescence was displayed onthe X-axis and the log of PI fluorescence was displayed on the Y axis.The data was analyzed by the CellQuest program (Becton Dickinson). Foreach analysis, 10,000 cells events were recorded.

Example 4 Cell Cycle

Cells were pelleted by centrifugation, and washed twice with PBS, fixedwith 70% (v/v) cold ethanol (−20° C.), and stored at 4° C. for at least24 hours. The cells were washed in PBS. Cell pellets were stained withPI/RNase staining solution. The cell suspension was incubated in thedark at room temperature for 30 min. DNA content was determined using aFACSCalibur flow cytometry (Becton Dickinson, Mount View, Calif.).Percentages of cells in Sub-G1, G₁, S and G₂/M stages of the cell cyclewere determined with DNA histogram-fitting program (Becton Dickinson). Aminimum of 10,000 events per sample was recorded.

Example 5 Synthesis of Aza-C-5′-petroselinic Acid Ester

Petroselinic acid (1.75 mmol, 494 mg) was dissolved in toluene (3 ml).DMF (10 μl) was added, followed by oxalyl chloride (3.6 mmol, 457 mg)over 10 min at room temperature. After 3 h, the toluene was removed invacuo.

Aza-C (1.57 mmol, 427 mg) was suspended in DMA (6 ml), HCl (1 M in Et₂O,2.0 mmol, 2.0 ml) was added, and after 5 min at room temperature theEt₂O was removed in vacuo. The resulting turbid solution was cooled inan ice-water bath, and the acid chloride, dissolved in DMA (2 ml), wasadded over 40 min. The reaction mixture was stirred overnight while thetemperature slowly reached room temperature. After 24 h, the solventswere removed at ca. 0.1 mbar. The residue was partitioned betweensaturated. aq. NaHCO₃, and EtOAc (25 ml of each). The aqueous phase wasextracted with another 3×25 ml EtOAc. The organic phases were combined,washed with brine, and dried (MgSO₄). After removal of the solvents invacuo, the crude product (600 mg) was purified by flash chromatography(SiO₂, CH₂Cl₂ with 2.5, 5, and 10% MeOH). Finally, the product was driedat ca. 0.25 mbar overnight. Yield: 210 mg (24%).

Example 6 Synthesis of Aza-C-5′-petroselaidic Acid Ester

Petroselaidic acid (1.77 mmol, 500 mg) was dissolved in toluene (3 ml).DMF (10 μl) was added, followed by oxalyl chloride (3.6 mmol, 457 mg)over 10 min at room temperature. After 3 h, the toluene was removed invacuo.

Aza-C (1.75 mmol, 427 mg) was suspended in DMA (6 ml), HCl (1 M in Et₂O,2.0 mmol, 2.0 ml) was added, and, after 5 min at room temperature, theEt₂O was removed in vacuo. The resulting turbid solution was cooled inan ice-water bath, and the acid chloride, dissolved in DMA (2 ml), wasadded over 2 h. The reaction mixture was stirred overnight while thetemperature slowly reached room temperature, then it was heated at 30°C. for 2 h. After cooling to room temperature, the reaction mixture waspartitioned between saturated. aq. NaHCO₃, and EtOAc (25 ml of each).The aqueous phase was extracted with another 3×25 ml EtOAc. The organicphases were combined, washed with water, and brine, and dried (MgSO₄).After removal of the solvents in vacuo, the ester was obtained as awhite powder. Yield: 500 mg.

Example 7 Metabolic Stability of 5-Aza-5′-Petroselinic Acid Ester inPooled Human Plasma

5-Aza-C-5′-petroselinic acid ester was spiked into pooled human plasmaat five concentration levels (0.1, 1, 3, 10, and 30 μM, respectively).The mixture was incubated in a shaking water batch at 37° C. Aliquots(100 μl) of the incubation solutions were withdrawn in triplicate (n=3)at the designed incubation period (0, 15, 30, 60, and 120 minutes), andplasma protein was immediately precipitated using acetonitrilecontaining 0.1% formic acid (3001). Negative controls were prepared withthe test compound and Aza-C in the assay buffer (PBS, pH 7.4) at oneconcentration of incubation (1 μM). After centrifugation, thesupernatant was directly introduced for LC-MS-MS analysis. See Table 1.

TABLE 1 Concentration % Remaining of initial (mean ± SD, n = 3)Half-Life (μM) 0 min 15 min 30 min 60 min 120 min (min) 0.1 100 95.0 ±2.5 92.9 ± 2.0 83.7 ± 2.5 51.3 ± 1.2 125 1 100 96.3 ± 5.3 90.9 ± 2.579.9 ± 3.2 46.3 ± 2.2 107 3 100 97.5 ± 3.3 91.5 ± 3.6 83.6 ± 1.3 50.9 ±0.7 122 10 100 97.3 ± 1.1 91.4 ± 0.6 78.6 ± 1.8 45.7 ± 0.6 104 30 10093.6 ± 1.3 85.9 ± 2.9 71.7 ± 2.3 40.6 ± 0.5 91

Example 8 Cytotoxicity of Aza-C and 5-Aza-C-5′-Petroselinic Acid

The cytotoxicity of Aza-C and 5-Aza-C-5′-petroselinic acid wasdetermined in a breast cancer cell line MT-3 and the adriablastinresistant cell line MT-3/ADR. The MT-3/ADR overexpress theMDR-1/p-glycoprotein. The cells were seeded in 96-well plates with 5×10³cells per well, in RPMI 1640 medium with 2 mM glutamine and 10% FBS. Thecells were incubated for 24 hours. The test compounds were dissolved inDMSO and further diluted in medium just prior to use. 6 wells were usedper test concentration. The cells were incubated with test compound for24 hours. 20 μl of freshly prepared MTT solution was added to each welland incubated for 4 hours. IC50 values were determined from growthcurves presented graphically based on 8 different concentrations rangingfrom 0.01 μM to 100 μM. The results are presented in Table 1. Similaractivity was obtained for Aza-C and 5-Aza-C-5′-petroselinic acid in theMT-3 breast carcinoma cell line, but in the MT-3/ADR resistant cell linethe activity of Aza-C was lost. No activity was observed in theconcentration range tested up to 100 μM, whilst 5-Aza-C-5′-petroselinicacid remained active with a similar IC50 value in the resistant cellline versus the non-resistant MT-3 line. See Table 2.

TABLE 2 Cytotoxic activity of Aza-C and 5-Aza-C-5′-petroselinic acid inbreast carcinoma with or without the expression of multi drugresistance. Aza-C 5-Aza-C-5′-petroselinic IC50 (μM) acid IC50(μM) MT-3breast carcinoma 12.62 ± 2.35 12.32 ± 6.37 MT-3/ADR resistant >100 12.02± 8.30 breast carcinoma

Example 9 Antiproliferative Activity of Aza-C and5-Aza-C-5′-Petroselinic Acid

The antiproliferative activity of Aza-C and 5-Aza-C-5′-petroselinic acidwas determined in the Hela mutant cervix carcinoma cell line at 24 and72 hours exposure. The cells were seeded in 96-well plates with 5×10³cells per well, in RPMI 1640 medium with 2 mM glutamine and 10% FBS. Thecells were incubated for 24 and 72 hours. The test compounds weredissolved in DMSO and further diluted in medium just prior to use. 6wells were used per test concentration. The cytotoxicity was determinedusing the MTT assay, 20 μl of freshly prepared MTT solution was added toeach well and incubated for 4 hours. IC50 values were determined fromgrowth curves presented graphically based on 8 different concentrationsranging from 0.01 μM to 100 μM. Similar cytotoxic activity was obtainedwith prolonged exposure for 72 hours for the two compounds, butsurprisingly the cytotoxic effect for 5-Aza-C-5′-petroselinic acid wasalready present after 24 hours exposure. A different time profile isobserved for Aza-C and 5-Aza-C-5′-petroselinic acid, with a rapid onsetof cytotoxic effect for 5-Aza-C-5′-petroselinic acid. See FIG. 1.

Example 10 Impact of Nucleoside Transporter Inhibition on CytotoxicActivity in Carcinoma Cells for Aza-C and 5-Aza-C-5′-Petroselinic Acid

The impact of nucleoside transporter inhibition on cytotoxic activityhas been evaluated in Hela mutant cervix carcinoma cells for Aza-C and5-Aza-C-5′-petroselinic acid. Dipyridamole was used as an inhibitor ofthe equilibrative nucleoside transporters hENT1 and hENT2. The cellswere seeded in 96-well plates with 5×10³ cells per well, in RPMI 1640medium with 2 mM glutamine and 10% FBS. The cells were pre-incubated for24 hours. Dipyridamole (10 μM) was added to the cells 30 minutes priorto the addition of the test compounds. The test compounds were dissolvedin DMSO and further diluted in medium just prior to use. 6 wells wereused per test concentration. The cells were incubated with test compoundfor 72 hours. 20 μl of freshly prepared MTT solution was added to eachwell and incubated for 4 hours. IC50 values were determined from growthcurves presented graphically based on 8 different concentrations rangingfrom 0.01 M to 100 μM. The results are presented in Table 3. Theactivity of Aza-C was reduced 3 fold by the addition of the nucleosidetransport inhibitor Dipyridamole, indicating that influx and efflux ofAza-C in the Hela cells are partly dependent on the nucleosidetransporters hENT1 and hENT2. The cytotoxic activity of5-Aza-C-5′-petroselinic acid was not only maintained but increased10-fold when the hENT1 and hENT2 nucleoside transporters were blocked bythe use of dipyridamole. This may be of particular importance inpatients where the activity of Aza-C is not present due to lack ofexpression of nucleoside transporters. See Table 3.

TABLE 3 Cytotoxic activity of Aza-C and 5-Aza-C-5′-petroselinic acid inHela cervix carcinoma cells with or without nucleoside transportinhibitor dipyridamole. 5-Aza-C-5′- Azacytidine petroselinic acid IC50(μM) IC50 (μM) Hela 4.32 4.74 Hela with 12.77 0.42 dipyridamole

Example 11 Gene Expression of Estrogen Receptor β (ERβ) in Breast CancerCell Lines After Treatment with Azacytidine or 5-Aza-C-5′-PetroselinicAcid

The gene expression (determined on RNA level) of estrogen receptor betawas determined by quantitative real-time PCR. (TaqMan). MCF-7 mammarycarcinoma cells were grown in estrogen deficient media (Phenol-Red-freeRPMI with 2% glutamine and 10% charcoal-dextran treated fetal calfserum). The cells were seeded into 25 cm² flasks and attached for 24hours prior to treatment with 1 μM of azacytidine or5-Aza-C-5′-petroselinic acid. One untreated control was included ascontrol. The cells were harvested after 5 days of exposure to thecompounds, they were harvested by trypsination, washed, and shock frozenin liquid nitrogen.

The total RNA was extracted from approximately 10⁶ shock frozen MCF-7cells, the RNA concentration and purity was measured, RNA wastranscribed into cDNA using TaqMan Reverse Trancription reagents(N808-0234). Real-time quantification was performed using standardprotocols and premixed PCR reagents. The primer-probe mixes were orderedfrom Applied Biosystems, ER β (ID Hs00230957_ml) and housekeeping genehydrocylmethyl-bilane synthase HMBS (ID Hs00609297_ml). Gene expressionwas calculated using the comparative delta-delta C_(t) method. Theinduction of expression of ER β was 4.14 fold after exposure to5-Aza-C-5′-petroselinic acid compared to only 2.51 fold after exposureto azacytidine, see Table 4. This may be of high relevance in hormonerefractory tumors where hormone sensitivity can be restored. See Table4.

TABLE 4 x-fold induction of Ct ER β Ct HMBS delta Delta delta the ERβgene Untreated 35.36 25.91 9.45 1 μM 32.97 25.92 7.06 −2.40 5.265′Aza-C-5′- elaidic acid 1 μM aza-C 34.31 26.18 8.13 −1.32 2.51

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A compound according to Formula (I)

wherein, R is H, R₅C(O), R₅CH₂OC(O), or R₅CH₂NHC(O); R₁ is

where the crossing dashed line illustrates the bond formed joining R₁ tothe molecule of Formula (I); R₂ and R₃ are independently OH or H,provided that R₂ and R₃ are not simultaneously OH; R₄ is H, R₅C(O),R₅CH₂OC(O), or R₅CH₂NHC(O), provided that R and R₄ are notsimultaneously H; R₅ has the general formula:CH₃—(CH₂)_(n)—(CH═CH—CH₂)_(m)—CH═CH—(CH₂)_(k)—; k is 4; m is 0 or 1; andn is an integer from 0 to 10, or a pharmaceutical salt thereof.
 2. Thecompound according to claim 1, wherein n is
 10. 3. The compoundaccording to claim 1, wherein R₁ is


4. The compound according to claim 3, wherein R₄ is H.
 5. The compoundaccording to claim 1, wherein R is R₅C(O), R₁ is

R₂ is H, R₃ is OH, R₄ is H, k is 4, m is 0, and n is
 10. 6. Apharmaceutical composition comprising: the compound of claim 1 and apharmaceutical excipient, diluent, and/or carrier.
 7. A method oftreating a subject for a neoplastic condition, said method comprising:selecting a subject with a neoplastic condition and administering to thesubject a compound of the formula:

wherein, R is H, R₅C(O), R₅CH₂OC(O), or R₅CH₂NHC(O); R₁ is

 where the crossing dashed line illustrates the bond formed joining R₁to the molecule of Formula (I); R₂ and R₃ are independently OH or H,provided that R₂ and R₃ are not simultaneously OH; R₄ is H, R₅C(O),R₅CH₂OC(O), or R₅CH₂NHC(O), provided that R and R₄ are notsimultaneously H; and R₅ has the general formula:CH₃—(CH₂)_(n)—(CH═CH—CH₂)_(m)—CH═CH—(CH₂)_(k)—; k is 4; m is 0 or 1; andn is an integer from 0 to 10, or a pharmaceutical salt thereof, underconditions effective to treat the neoplastic condition in the subject.8. The method of claim 7, wherein the neoplastic condition is acancerous disease.
 9. The method of claim 8, wherein the cancerousdisease is a solid tumor or a hematological cancer or malignancy. 10.The method of claim 8, wherein the cancerous disease is leukemia,lymphoma, multiple myeloma, or myelodysplastic syndrome.
 11. The methodof claim 9, wherein the solid tumor is a cancer of a tissue selectedfrom the group consisting of breast, ovary, prostate, brain, bladder,and lung.
 12. The method according to claim 7, wherein n is
 10. 13. Themethod according to claim 7, wherein R₁ is


14. The method according to claim 13, wherein R₄ is H.
 15. The methodaccording to claim 7, wherein R is R₅C(O), R₁ is

R₂ is H, R₃ is OH, R₄ is H, k is 4, m is 0, and n is
 10. 16. A method oftreating a subject for an inflammatory condition, said method,comprising: selecting a subject with an inflammatory condition andadministering to the subject a compound of the formula:

wherein, R is H, R₅C(O), R₅CH₂OC(O), or R₅CH₂NHC(O); R₁ is

 where the crossing dashed line illustrates the bond formed joining R₁to the molecule of Formula (I); R₂ and R₃ are independently OH or H,provided that R₂ and R₃ are not simultaneously OH; R₄ is H, R₅C(O),R₅CH₂OC(O), or R₅CH₂NHC(O), provided that R and R₄ are notsimultaneously H; and R₅ is a C₃-C₂₆ alkenyl, wherein R₅ has the generalformula:CH₃—(CH₂)_(n)—(CH═CH—CH₂)_(m)—CH═CH—(CH₂)_(k)—; k is 4; m is 0 or 1; andn is an integer from 0 to 10, or a pharmaceutical salt thereof, underconditions effective to treat the inflammatory condition in the subject.17. The method of claim 16, wherein the inflammatory condition is aninflammatory state of the lung, connective tissue, gastro-intestinaltract, or vasculature.
 18. The method according to claim 16, wherein nis
 10. 19. The method according to claim 16, wherein R₁ is


20. The method according to claim 19, wherein R₄ is H.
 21. The methodaccording to claim 16, wherein R is R₅C(O), R₁ is

R₂ is H, R₃ is OH, R₄ is H, k is 4, m is 0, and n is 10.